Antibiotic 21,190 rp and process for preparing same

ABSTRACT

THE NEW ANTIBIOTIC 21,190 RP IS PREPARED BY CULTIVATING THE HITHERTO UNKNOWN MICROORGANISM STREPTOMYCES HYGROSCOPICUS DS 23,230 (NRRL 3576) UNDER AEROBIC CONDITIONS IN AN AQUEOUS NUTRIENT MEDIUM. THE ANTIBIOTIC IS ACTIVE AGAINST GRAM-POSITIVE AND SOME GRAM-NEGATIVE MICROORGANISMS. IT CAN BE USED AS A GROWTH PROMOTIN AGENT FOR ANIMALS.

July 2, 197 MANCY ET N T fiillk ANTIBIOTIC-21,19O RP AND PROCESS FORPREPARING SAME Filed Nov. 2'7, 1972 2 Sheets-Sheet 1 Bil WAVE NUMBER (cwQ0 I L I 1 .1

WAVELENG7H(NA NOMETRES) 2 Sheets-Sheet g a ZQQQEM IR wzm Qw $3 mm 3 mm 9Q m m D. MANCY ET July 2, H@?@ ANTIBIOTIC-21,19O R? AND PROCESS FORPREPARING SAME Filed NOV. 27, 1972 Q8 SQ 8? 33 89 United States PatentOfiice 3,822,350 Patented July 2, 1974 ABSTRACT OF THE DISCLOSURE Thenew antibiotic 21,190 RP is prepared by cultivating the hitherto unknownmicroorganism Streptomyces hygroscopicus DS 23,230 (NRRL 3576) underaerobic conditions in an aqueous nutrient medium. The antibiotic isactive against Gram-positive and some Gram-negative microorganisms. Itcan be used as a growth promoting agent for animals.

This invention relates to a new antibiotic, hereinafter denoted by thenumber 21,190 RP, to its preparation by culture of a Streptomyces strainidentified more completely hereinafter and denoted by the nameStreptomyces hygroscopicus DS 23,230 and to compositions containing theantibiotic.

A specimen of Streptomyces hygroscopic-us DS 23,230 has been depositedat the US. Department of Agriculture, Northern Regional ResearchLaboratory, at Peoria, 111., U.S.A., and has been given the number NRRL3576; a sample of the microogranism can be obtained from theaforementioned Research Laboratory.

Antibiotic 21,190 RP is of value because of the antimicrobial activitywhich it exerts principally against Grampositive microorganisms, inparticular against staphylococci, as well as against certainGram-negative microorganisms, especially against bacteria of theNeisseria genus, in conjunction with its low toxicity.

Antibiotic 21,190 RP is characterised by the following physico-chemicalproperties:

Appearance: white crystalline powder.

Elementary analysis: it contains carbon, hydrogen, oxygen and chlorinein the following proportions: C=50.5 H=6.6%; O=38.4%; and Cl=4.5%

Solubility: it is soluble in water at pH 9 (10 g./l.) and indimethylformamide (50 g./l.), sparingly soluble in methanol and ethanoland practically insoluble in hexane and benzene.

Melting point: (determination on a Kofler block) 226-228 C.

Ultra-violet spectrum: when dissolved in aqueous 0.1 N sodium hydroxidesolution, 21,190 RP shows an absorption maximum at 292 nm.

( lia; 5

and an absorption minimum at 257 nm.

( lfin;

FIG. 1 of the accompanying drawings shows the U.V. spectrum of 21,190 RPdissolved in aqueous 0.1 N sodium hydroxide solution at a concentrationof 50 ag/cc.

Infra-red spectrum: (determination from tablets of a mixture with KBr)this spectrum is shown in FIG. 2, in which the abscissae give thewavelength expressed in miccrons (lower scale) and the wave number incm.- (upper scale) and the ordinate gives the optical density.

The principal infra-red absorption bands of 21,190 RP, expressed in Wavenumbers (cmf are given in Table I.

TABLE I 3,440 S (partially due 1,060 S to H 0) 1,035 vS 2,970 m 975 m2,930 S 942m 2,900 sh 920 sh 2,840 sh 900 w 1,735 S 865 m 1,715 m 848 w1,630m (H 0) 828 w 1,570 m 815 w 1,450 S 775 m 1,403 m 730 m 1,3808 690w1,350m 678 vw 1,335 sh 655 w 1,305 sh 630 sh 1,248 S 612w 1,220 w 570 w1,192 m 548 W 1,165 m 525 vw 1,122 s 510 vw 1,090 S where vS=verystrong; S=strong; m=medium; w=weak; vw=very weak; sh=shoulder.

Optical rotation:

Colour reactions: antibiotic 21,190 RP gives the following reactions:Positive with the following reagents: cysteine-carbazole, sulphuricacid, Wheeler-Tollens, Bial, Fehling (after acid or alkaline hydrolysis)and Molisch. Negative with Millon, Nessler, Ehrlich, ZimmermanBitto,Sakaguchi, Elson-Morgan and Dische reagents.

Chromatographic migrations: The Rf-values for 21,190 RP, deposited in anamount of 100 ,ug. on a plate of Kieselgel H (Merck) and revealed bybioautography with Staphylococcus aureus 209 P (ATCC 6538 P) as thesensitive microorganism, or by spraying with sulphuric acid and heatingat 100 C., are given in Table II for some solvent systems.

TABLE II Solvents: Rf

Chloroform 0 Benzene 0 Benzene-methanol 90-10 (v./v.) 0.15Benzene-methanol -20 (v./v.) 0.60 Benzene-methanol 50-50 (v./v.) 0.87Acetone 0.80 Methanol 0.85

ANTIBIOTIC ACTIVITY AND TOXICITY (A) Bacteriostatic activity in vitro of21,190 RP 3 TABLE 111 strain (Institut Pasteur) 0.25 Diplococcuspueumoniae, Til strain (Institut Pasteur) 0.1 Neissseria catarrhalis (A152, Institut Pasteur) 1.25 Neissseria meningitidis (5813, InstitutPasteur) 0.6 Neissseria gonorrhoeae (A 50, Institut Pasteur) 1.25Bacillus subtilis-ATCC 6633 15 Bacillus cereus-ATCC 6630 9 MycobacteriumspeciesATCC 607 150 Escherichia cli--ATCC 9637 150 Shigella dysenteriae,Shiga L (Institut Pasteur) 150 Salmonella paratyphi A (Lacasse strain,In-

stitut Pasteur) 150 Salmonella schottmuelleri (paratyphi B) Fougencstrain (Institut Pasteur) 150 Proteus vulgaris 150 Klebsiellapneum0niaeATCC 10.031 150 Pseudomonas aeruginosa (Bass strain, InstitutPasteur) 150 Brucella bronchz'septica (CN 387Wellcome Institute) 35Pasteurella multocida (A 125, Institut Pasteur) 0.2 Mycoplasmagallisepticum (A 5.14, Institut Pasteur) 3 21,190 RP also possesses amarked bactericidal activity, in vitro, against staphylococcus; theratio between the "bacteriostatic and bactericidal concentrations is ofthe same order as that of penicillin G. (B) Toxicity The toxicity of21,190 RF is low as is shown by the results obtained by subcutaneousadministration to mice:

LD ==2,500 mg./kg. animal body weight administered subcutaneously.

LD =1,000 mg./kg. animal body weight administered subcutaneously.

(C) Antimicrobial activity in vivo Antibiotic 21,190 RP is active, inmice, against experimentally-induced infections caused by staphylococci,steptococci and meningococci, when it is administered subcutaneously,but is inactive when administered orally. When the product isadministered subcutaneously for two consecutive days to mice, the 50%curative doses (CD are between 2 and 18 mg./kg. animal body weight,administered subcutaneously, depending on the bacterium used.

The organism which produces antibiotic 21,190 RP is 'a strain ofmicroorganism which has been isolated from -a sample of earth taken inGreat Britain, in Middlesex,

and to which the number DS 23,230 (NRRL 3576) has been given.

The isolation of this microorganism was carried out by following thegeneral method which consists of suspending a small amount of earth insterile distilled water, diluting the suspension to dilferentconcentrations, and spreading a small-volume of each dilution on thesurface of Petri dishes containing a nutrient agar medium. Afterincubation for several days at 26 C., which makes it possible for themicroorganisms to develop, the colonies which it is desired to isolatein order to continue the investigation of them are removed andtransplanted to sloping nutrient agars for the purpose of producing moreabundant cultures of them.

The strain of microorganism DS 23,230 belongs to the Streptomyces genusand, more precisely, is related to the species Streptomyceshygroscopicus, the essential characteristics of which have been definedby H. D. Tresner and E. J. Backus (Applied Microbiology, 4, 243-250,1956) and by S. A. Waksman (The Actionomycetes, II, The Williams andWilkins Company, Baltimore, 1961, pages 230-231). This is why it hasbeen called Streptomyces hygroscopicus, DS 23,230.

S. hygroscopicus, DS 23,230 possesses, in effect, the following threeproperties which correspond to the three characteristics by which H. D.Tresner and E. J. Backus as well as S. A. Waksman define the species S.hygroscopicus: (a) its sporiferous filaments generally terminate inclosed spirals with a coil of a few turns; these sporiferous filamentsare usually inserted along a main filament, forming clusters which maybe more or less elongated; (b) when its sporulated aerial mycelium hasreached a good stage of development, it shows a dark grey colourationcorresponding to that shown by the species S. hygroscopicus, and (c) oncertain culture media which make good sporulation possible, after aging,shiny black regions with a wet appearance appear in the sporulatedsurfaces, characteristic of the species S. hygroscopicus; in the case ofS. hygroscopicus, DS 23,230, the conversion of the dark green sporulatedaerial mycelium into a black coating takes place only in quite aninconspicuous way, which is in general limited to small points or smallregions distributed over the sporulated surface rather than taking placeover this entire surface area. The appearance of these black regions ishowever evident and can be observed in particular on Hickey and Tresneragar, Pridham yeast extract agar, Pridham oatmeal and tomato, agar,glucose-asparagine agar, starch-nitrate agar and Pridhamstarch-inorganic salts agar.

H. D. Tresner and E. J. Backus describe the production of awine-coloured soluble pigment, on certain media, varying according tothe particular case, by certain strains of the species S. hygroscopicus.The strain DS 23,230 possesses this property to a very marked extent; itproduces a reddish pink soluble pigment which may be more or lessviolet-coloured on quite a large number of media, and in such a way thatin many cases it is capable of colouring the aerial mycelium which thenassumes a pink shade before showing the characteristic gfey colourationwhich it has when sporulation takes p ace.

On the few culture media where its morphological appearance isdescribed, the strain S. hygroscopicus mentioned as reference by S. A.Waksman in The Actinomycetes shows a certain number of differencescompared with the strain DS 23,230. The most noticeable are that it doesnot give any soluble pigment on gelatine, that it produces a lightyellow soluble pigment on glucoseasparagine agar, and that it gives awell developed culture on nitrate agar containing sucrose, whilst thestrain DS 23,230 gives a dark orange-brown soluble pigment on gelatine,as well as on asparagine-glucose agar and, due to the fact that it doesnot utilise sucrose, does not develop on Czapek synthetic nitrate agarcontaining sucrose. Furthermore, in no case is it stated that itsnonsporulated aerial mycelium is coloured pink, as has just been statedin the case of the strain DS 23,230. However, these several dilferencesare not sufficiently important, according to the ideas of H. D. Tresnerand E. J. Backus, for it to be considered that the strain DS 23,230 canconstitute a species which is different from the species S.hygroscopicus, since it presents the main characteristics of S.hygroscopicus which serve to define it.

S. hygroscopicus, DS 23,230 forms sporiferous filaments which generallyterminate in closed spirals containing 1 to 5 turns, although veryoccasionally spirals forming a larger number of turns, or somesporiferous filaments which are simply curved over at their end part 6Ref. DYeast Extract AgarT. G. Pridham et coll- Antibiotics Annual,1956-57, p. 950 Ref. E-Tomato Paste Oatmeal AgarT. G. Pridham etcollAntibiotics Annual, 1956-57, p. 950 Ref. F-Peptone 0.5 %-rneatextract 0.3 %--tyrosine Without forming a complete turn, or sometimesspirals 5 0.5%agar 2% which may be more or less loose and unwound, areRef. GMelanin formation medium-The Actinoobserved. The sporiferousapparatus possesses a cluster mycetes, vol. 2, p. 333-No. 42-S. A.Waksman-- structure, the spiral sporiferous filaments, which them- TheWilliams and Wilkins Company, Baltimore, 1961 selves can show somebranches, being inserted along 10 Ref. H-W. E. Grundy et c0llAntibioticsand Chem, a main filament which can be quite long. The spores are 2,401, 1952 oval and measure 0.6 to 0.8/1.0 to 1-2/L. Microscopic Ref.I-Inorganic Salts-Starch Agar-T. G. Pridham examinations have shown anidentical arrangement of et call-Antibiotics Annual, 195657, p. 951 thesporiferous apparatus on Hickey and Tresner agar Ref. JCorresponds tothe formulation W-l, wherein and on Pridham starch-inorganic salts agar.30 g. of sucrose are replaced by 15 g. of glucose The culturecharacteristics and the biochemical prop- Ref. K-Corresponds to theformulation W-l, wherein erties of S. hygroscopic-us, DlS' 23,230 aregiven in Table 30 g. of sucrose are replaced by 15 g. of glycerine 1Vwhich follows. Unless otherwise stated, they are those Ref. LPlaingelatm--prepared according to the inof cultures which have reached agood stage of developstructions 1n Manual of Methods for Pure Culturement, having been aged for about 3 to 4 weeks at 26 Study of Bacteria ofthe Society of American Bac- C. These properties have been observed onnutrient teriologists-Geneva, N.Y., II -l8 agars and broths usuallyemployed to determine the Ref. MManual of Methods for Pure Culture Studymorphological properties of strains of Streptomyces, the of Bacteria ofthe Society of American Bacteriolocultures on agar media being carriedout on agar slopes. g1sts-Geneva, N.Y., II -18 A certain number ofculture media used were prepared Ref. N-Synt-hetic medium ofDimmick-(not containin accordance with formulations indicatedin TheActinoing agar)Manual of Methods for Pure Culture mycetes (S. A.Waksman, pp. 193-197, Chronica Bo- Study of Bacteria of the Society ofAmerican Bacteritanica Company, Waltham, Mass., U.S.A., 1950); inologists, -e 50- this case, they are indicated by the letter W followedby Ref. OC rr p n s to f rm n W in the number which was given them inThe Actinomy- 30 g. of sucrose are replaced by 15 g. of glucose cetes.The references or constitutions of the other culture P0rf6SP0I1dS t0 theformulatloll wherein media are as follows: the sucrose is omitted andreplaced by small strips of filter paper partially immersed in theliquid I 7 9, 7, 1 i g i Pndham Ref. Q-Commercially available skimmedmilk powder, 8 C0 n1 10.165 nnua reconstituted in accordance with themanufacturer's Ref. BFormulat1on W-23, to which 2% of agar hasinstructions been added Ref. R-Medium indicated for the research of theproducof Bacteflologyi 142, tion of H 8 by: H. D. Tresner and F.Danga-Journal 1949 of Bacteriology, 76, 239-244, 1958.

TABLE IV Vegetative mycellum (V.m.) Aerial apparatus (comprising theDegree of or underside of combination of the aerialmycelium SolubleObservations and biochemical Culture media development the culture andthe sporulation) pigment properties Hickey and Tresner Good Undersidelight Pink to pink-grey and dark grey, Light orange- Sporiferousapparatus in clusters. agar (Ref. A). orange-brown. with points havingthe black brown. .Sporiferous filaments ending in appearancecharacteristic of closed spirals oil to 5 turns. Oval "Hygroscopicus".1srores measuring 0.6 to 0.8/1.0 to Ergerson agar (Ref. Very good"...Urdersideorange- Pink-white Orange-brown... I

IOWIl. Bennett agar (Ref. 0) ..do Underside dark Light pink-grey doorange-brown. Pridham yeast extract do ..d0 Greyish-pink to light greyand Very dark agar (Ref. D). dark grey, with points having orange theblack appearance characbrown.- teristic of Hygroscopicus". Pridhamoatmeal and do Underside black- Greyish-pink to grey with many Very darktomato agar (Ref. brown. points having the black appearorange- E). ancecharacteristic 0! Hygrobrown, rangscopicus. ing towards blackish.Glncose-peptone agar Good Underslde violet- Light greyish-pink to lightviolet- Slightly violet- (W-7). brown. grey. erploured X'OWD- Nutrientagar (W5).. Moderate Underside yellow. Greyish-white. Poorly developed"None Nutrient agar contain- Quite good- Underside light whitish Lightyellow- Solubilisation of tyrosine: positive: ing tyrosine (Ref. F).brown-yellow. brown. Tyrosine-yeast extract .-do Underside Greyish-whiteVery light Production of melanin: negative agar[Melaninforyellowishbrownish: (readings taken according to the mation medium ofwhite. author's recommendations). Waksman] (Ref. G). Krainsky calciumExtremely V.m. colourless None None Solubllisatlon of the malate: nonemalate agar (Ref. H). poor. to whitish, in or extremely low.

trace amounts. Ovalbumin agar (W-12)- Very mod- V.m. orange- .do0range-brown 8! 6. Town. Glucose-asparagine Quite good Underside darkLight pink-grey to light grey and Very dark agar W-2 brown. dark grey,with a few small orangepoints having the black appearbrown. aucecharacteristic of Hygroscopicus Glycerine-asparaglne Good Underside veryGreyish-orange to light grey. Blackish-brown.

agar (W-3). dark brown. smglltiiroplets oi yellow-brown 9X11 8. on.Starch-nitrate agar Very moder- Underside very Pink-white toviolet-grey, with a Light pink- Hydrolysis of the starch: positive.

(W-lO). ate dark violet. few small points having the violet.

black appearance characteristic of Hygroscopicus.

TABLE IVContinued Vegetative mycelium (V.rn.) Aerial apparatus(comprising the Degree of or underside of combination of the aerialmycclium Soluble Observations and biochemical Culture media developmentthe culture and the sporulation) pigment properties Pridham starch- Verygood Underside dark Greyisli-white to light grey and Greyish-yellow-Sporiferous apparatus in clusters. inorganic salts agar yellow-brown.dark grey, with points having brown. Sporlierous filaments ending in theblack appearance characterclosed spirals of 1 to 5 turns. Oval istic ofHygroscopicus". spores measuring 0.6 to 0.8/1.0 to 1.2 Hydrolysis of thestarch: positive. Czapek synthetic agar Practically None cgi itfiiningsucrose none. Czapek synthetic agar Good Underside very Greyisli-pinkviolet. Exudation Very dark Cgltilllhlg glucose diirlktreddishof a fewsmall pink droplets. reddish-violet.

e v o e Czapek synthetic agar Very good Underside very Violet-pink.Exudation oi small Very dark containing glycerine dark red-brown. pinkdroplets. reddish- (Ref. K). violetbrown. Potato culture (W-27) ..doV.rn. very well Greyish-white tinged light pink Orange-brown developedand in places. Numerous droplets of to dark violetvery wrinkled; lightbrownish-yellow to orangereddish-brown. completely brown exudation.covered by the aerial mycelium. 12% pure gelatine Good Well developedwhitish. Moderately developed-.. Orangebrown.-. Liquefaction oi thegelatine: quite (Ref. L). surface culture. rapid.

Underside light oran e-brown. Nutrient nitrate broth Moderate....-.Thin, rownishwhiti h. Very moderately devel- None Production ofnitrltcsfrom nitrates:

(Ref. M ellow ring. oped. negative. Starch nitrate broth W 'tish lingand ....-do.. Very weak Production ofnitn'tesirom nitrates:

(W-19). velum; brownish. positive. Dimmick glucose ni- Moderate Smallyellowishwhitish. In trace amounts.-. do Production of nitrites fromnitrates:

trate broth (Ref. N). brown colonies positive at the start of the eulatthe surface of ture, but quite quickly becomes the culture. negative.Czapek synthetic broth Moderate Small whitish Whitish. Very moderatelydevel- None containing glucose colonies clusoped.

Ref. 0). tered together at the surface of the culture. Czapek syntheticbroth None.. Utilisation of the cellulose: negative containing cellulose(Ref. 1). Skimmed milk (Ref. Q). Poor Peptonisation without coagulation;

pH unchanged in 1 month. Tresner and Danga Quite good-.-. V-m. lightNone Very light Production of H28: negative (readagar for investigationyellowish-brown. yellowishings taken according to the of the productionof brown. author's recommendations). HzS (Ref. R).

The capacity of Streptomyces hygroscopicus, DS 23,230 for using varioussources of carbon and nitrogen to achieve its development has beendetermined according to the principle of the Pridham and Gottlieb method(J. of Each, 56, 107-114, 1948); the degree of development was observed,after a suitable incubation period at 26 C., on the base mediumindicated by the authors, by replacing either the glucose by the varioussources of carbon respectively tested, or (NH4)2S04 by the varioussources of nitrogen respectively tested. The results are given in TableV.

TABLE V Sources of Sources of carbon tested Utilisation nitrogen testedUtilisation D positive positive positive pos t ve negative positivenegative positive positive positive positive positive positive negativepositive Urea posit ve negative L-asparagine posit ve positive Glycinepositive positive Sarcosine negative negative DL-alanine positiveTrehalose positive DLvaline positive Cellobiose. negative DL-asparticacid positive Raiiinose positive L-glutamic acid... positive Dextrinpositive L-arginine positive Inulin. negative Starch positive Glycogenpositive Glycerol positive Erytliritol negative Adonitol negativeDulcltol negative DL-proline p D-morinitol... positiveL-hydroxyproline.-. positive D-sorbitol egative L-histidine positiveInositol pos ive Salicine negative I But weak.

According to a feature of the invention the antibiotic 21,190 RP isproduced by aerobically cultivating Streptomyces hygroscopicus DS 23,230(NRRL 3576), or a mutant thereof capable of producing the antibiotic,using an aqueous nutrient medium containing assimilable sources ofcarbon, nitrogen and inorganic substances, and isolating from the medium21,190 RP formed during the culture.

The culture of Streptomyces hygroscopicus DS 23,230 can be carried outby any of the known aerobic surface culture or submerged culturemethods, but the latter are preferred for reasons of convenience. Forthis purpose, the various types of apparatus which are currentlyemployed in the fermentation industry may be used. In particular, thefollowing sequence of operations can be adopted:

Streptomyces hygroscopicus, DS 23,230-stock culturc on agar culture inan agitated flask inoculum culture in a fcrmenter production culture ina fermenter The fermentation medium must contain an assimilable sourceof carbon and an assimilable source of nitrogen, inorganic substances,particularly chlorides, and optionally growth-promoting factors; allthese ingredients may be supplied as well-defined products or complexmixtures such as those found in biological products of various origins.

As sources of assimilable carbon, there may be used carbohydrates suchas glucose, maltose, dextrins, starch, or other carbon-, hydrogenandoxygen-containing substances such as sugar alcohols, e.g. glycerol ormannitol, or certain organic acids, e.g. lactic acid or citric acid.Certain animal or vegetable oils such as lard oil or soya bean oil maybe advantageously used instead of, or in admixture with, theaforementioned carbon-, hydrogenand oxygen-containing substances.

The suitable sources of assimilable nitrogen are extremely varied. Theycan be very simple chemical compounds such as inorganic or organicammonium salts, urea or certain amino acids. They can also be complexsubstances containing principally nitrogen in a protein form, such ascasein, lactalbumin, gluten and their hydrolysates, soya bean flour,peanut meal, fish meal, meat extract, yeast extract, distillers solublesor corn-steep liquor.

Amongst the inorganic substances, some may have a buffering orneutralizing effect such as the alkali metal or alkaline earth metalphosphates or the carbonates of calcium or magnesium. Others contributeto the ionic equilibrium necessary for the development of Streptomyceshygroscopicus, DS 23,230 and for the production of 21,190 RP, such asthe chlorides and sulphates of the alkali metals and alkaline earthmetals. Some of them act more especially as activators of the metabolismof Streptomyces hygroscopicus, DS 23,230, e.g. the salts of zinc,cobalt, iron, copper and manganese.

The pH of the fermentation medium at the start of the culture should bebetween 6.0 and 7.8 and preferably between 6.5 and 7.5. The optimumfermentation temperature is 25 30 C., but satisfactory production isachieved at temperatures between 23 and 33 C. The rate of aeration ofthe fermentation broth can vary Within quite wide limits, but it hasbeen found that aeration rates of 0.3 to 3 litres of air per litre ofbroth per minute are particularly suitable. The maximum yield ofantibiotic 21,190 RP is obtained after 2 to 8 days culture, but thisperiod depends predominantly on the medium used.

It can be seen from the foregoing that the general conditions for theculture of Streptomyces hygroscopicus, DS 23,230 for the production of21,190 RP can vary to quite a large extent and can be adapted for eachparticular requirement.

21,190 RP can be isolated from the fermentation broth in the followingmanner:

The antibiotic is extracted from the filtrate of the fermentation brothby water-immiscible solvents, such as aliphatic alcohols having at least4 carbon atoms (e.g. butanol), chlorinated hydrocarbons (e.g. methylenechloride), esters (e.g. ethyl acetate) and ketones (e.g. methyl isobutylketone). This operation can be carried out at a pH between 3 and 9, andpreferably at about pH 7.

After decantation, the crude antibiotic can be isolated from itssolution in the above-mentioned organic solvents by concentration ofthese solutions under reduced pressure, followed by precipitation by anon-solvent or a poor solvent, for the antibiotic, such as hexane, or byallowing to stand in a cold chamber.

The crude 21,190 RP can be purified by conventional methods such asrecrystallisation, chromatography on various adsorbing agents orcounter-current distribution.

It is advantageous to purify 21,190 RP by recrystallisation from ethanolafter treatment with activated charcoal, followed by recrystallisationfrom a mixture of dimethylformamide and water.

The following Example illustrates the invention.

In the following, the activity is always determined by means of themicrobiological diffusion method, using Staphylococcus aureus 209 P(ATCC 6538 P) as the sensitive microorganism, by comparison with asample of pure 21,190 RP taken as the standard and containing 1,000gJmg. This activity is expressed in ,ug./ cc. for solutions and inug/mg. for solid products.

EXAMPLE 1 A l70-litre fermenter is charged with:

Tap water, sufficient to make up to 110 litres.

The pH is adjusted to 7.20 with 10 N sodium hydroxide solution cc.). Themedium is sterilised by bubbling steam at 122 C. through it for 40minutes. After cooling, the volume of the broth is 120 litres and the pHis 6.60. The broth is inoculated with a culture (200 cc.) ofStreptomyces hygroscopicus, DS 23,230 in a shaken Erlenmeyer flask. Theculture is developed at 26 C. for 29 hours with agitation and aerationwith sterile air; it is then suitable for inoculating the productionculture.

The production culture is carried out in a 800-litre fermenter, chargedwith the following substances: Soya bean flour kg 6 Distillers solubleskg 10 Calcium carbonate kg.. 2 Cobalt chloride-6H O g 8 Tap water,sufficient to make up to 325 litres.

The pH is adjusted to 7.80 with 10 N sodium hydroxide solution (1,200cc.), and the medium is sterilised by bubbling steam at 122 C. for 40minutes. After cooling, the volume of the broth is 355 litres. It ismade up to 400 litres by adding an aqueous sterile solution (40 litres)containing hydrated glucose (8 kg.) and an aqueous sterile solution (5litres) containing ammonium sulphate (800 g.) The pH is then 6.80.

The broth is then inoculated with the inoculum culture (40 litres) fromthe -litre fermenter described above. The culture is carried out at 26C. for 64 hours with agitation using a motor rotating at 205 revolutionsper minute and aeration with sterile air (20 mfi/hour).

After 24 and 48 hours of culture, an aqueous sterile solution (5 litres)is added, containing hydrated glucose At the end of the operation, thepH of the culture is 7.50 and the volume of the broth is 410 litres. Theamount of 21,190 RP present is 240 ,ug./ cc.

The broth is introduced into a tank equipped with a stirrer, and afiltration aid (Clarcel DIC) (30 kg.) is then added. The suspension isfiltered on a filter press and the filter cake is washed with water (100litres). The filtrate, the volume of which is 430 litres, is extractedin two stages with butanol (240 litres), using a group of centrifuges incountercurrent. The flow rate of the filtrate is adjusted to litres/hourand the flow rate of the solvent to 100 litres/hour. The extract, thevolume of which is 250 litres, contains the major portion of theactivity. It is washed with water (25 litres). The washed extract has avolume of 225 litres. It is concentrated at 35 C. under reduced pressure(20 mm. Hg) to a volume of 2 litres.

The concentrate is left to stand in a cold chamber at +5 C. After 12hours at this temperature the precipitate which has formed is filteredoff, washed with butanol (500 cc.) and dried. Antibiotic 21,190 RP (230g.), the strength of which is 260 ug./mg., is thus obtained.

Crude 21,190 RP (137 g.), prepared under the above conditions, but ofstrength 216 ,ug./mg., is suspended in ethanol (3.5 litres). Thesuspension is heated under reflux for 30 minutes and then filteredwhilst hot. The insoluble material is discarded. The filtrate, to whichdecolourising charcoal (Darco G 60) (10 g.) has been added, is stirredwhilst heating under reflux for 15 minutes and then filtered using afiltration aid (10 g.).

The solution obtained is concentrated under reduced pressure to a volumeof 1 litre. The concentrate is placed in a cold chamber at +5 C. for 12hours. After this period, the crystals which have formed are filteredoff, washed with ethanol (50 cc.) and dried. Crystalline 21,190 RP (26.2g.) of which the strength is 920 g./ mg., is thus obtained.

The antibiotic (85.5 g.), prepared under the above conditions, isdissolved in dimethylformamide (4.25 litres). The solution is clarifiedby filtration and then introduced into a flask equipped with a stirrer.Water (3 litres) is run slowly into the clarified solution, whilstcontinuing the stirring. After the end of the addition, the

1 1 suspension of crystals is cooled to +10 C. The crystals are thenfiltered off, washed with a dimethylformamide/ water (1:1 v./v.) mixture(1 litre) and then re-suspended in water (1.5 lires). They are finallyfiltered off and dried for 24 hours at 40 C. under reduced pressure (1mm.

organic salts. These supplementary feedstufis can either be mixed withthe animals rations or consumed directly, and usually represent about to20% of the ration.

The premixes," which are used for preparing complete rations orsupplementary feedstuffs, usually contain Hg). Crystalline 21,190 RP(61.5 g.), of strength 1,000 5 0.051 to 1210% 1by wflight of antibiotic21,fl90 RP diluted ./m melting at 226-228 C., is thus obtained. wit a pysio ogica y innocuous carrier, or examp e, an Sinti antibiotic 21,190RP is particularly active against amount of food. They constitute aconvenient interstreptococci and staphylococci, it can advantageously bemediate which makes it easier to distribute the antibiotic used in thelocal treatment of mastitis, metritis and cutane- 21,190 RP evenly infeedstuffs. The premixes themselves ous infections of domestic animals.At a dose of 1 mg./ are generally produced from concentrates whichcontain kg. animal body weight (intramuscular) the antibiotic 99.9 to20% by weight of antibiotic 21,190 RP, to which showed good activity inchickens infected with Borrelia a PEYSiOflOgCgllY infrfiocfiiiouscarrier ort edlrble denaturanzs anseriJla res onsible for avianspirochaetes). suc as ee yestu s, avouring agen s, ispersing agen s Theprt sen z invention includes within its scope thera- 15 or agents whichprevent agglomeration, and fillers, have peutic compositions forveterinary use comprising 21,190 been added. A concentrate can contain,for example, by RF in association with a physiologically acceptablecarrier weight, 99% of antibiotic 21,190 RP with 0.1% of dyeand/or acompound which may itself be physiologically stulf and 0.9% ofantiagglomerating agent. active, for example an antimicrobial agent suchas an The concentrates and premixes are generally powders. antibioticwith a different antibacterial spectrum, or an The supfplemenaryfleedstuffsd and the colrlnplfete confiposite antifungal a cut. feedstus can e eit er pow ers or in t e orm o gran- For theraieuticapplication, antibiotic 21,190 RP can ules, prepared according to theusual techniques. In these be made up in any of the usual various formssuitable different compositions, antibiotic 21,190 RP can be in for themethod of administration envisaged, such as liquid the form of fine freeparticles or covered with a coating. formulations (suspensions, ordispersions, preferably pre- The antibiotic 21,190 RP is suitable foradministrapared at the time of use from a solid composition), paste tionto all animals and especially to fowls. formulations (creams andointments) and solid formula- The following Examples illustrate thisaspect of the tions (vaginal tablets, ovules and powders). Theproporinvention. tion of 21,190 RP may be varied according to the methodEXAMPLE 2 "eatqlent and of t W fmmulamnh a Chicks (SELAF 91s aredistributed in batteries at the formulanogprefpared lmmedlafely befineuse for rate of 14 per coop, each coop containing chicks of the muscularin ection can comprise a unit dose of 21,190 Same Sex As many young hensas young cocks are used RP and a (10.56 Sultable. for Vetennary for Thechicks are placed in warm batteries from the age of i an Somme szfhneSolution ,buffered 1 day and during the 4 weeks of the experiment. Thefeedwith phosphates to give a suspension containing 1% stuffs to betested are dispensed from day 2' by weight of antibiotic. A powder fordusting can contain The chicks are Weighed and the amount of feedstufi50% i f of Z a powder F consumed is determined after 2 and 4 weeks. Thegain dlspersed a hquld suitable i i i f y mlectllm in weight and theconsumption index is calculated after 2 can contain up to 99% by weightof antibiotic in associa- Weeks and after 4 weeks. tion with aphysiologiqany acceptable dyestufi Such as The chicks receive a basicfeedstuff having the follow- Blue F.C.F. (Alphazurine FG-Food Blue No.2C.I. ing composition: No. 42,090) and with a physiologically acceptabledispers- Percent agent olntment Contau} to Flour of ereal by r d t eightof antibiotic, the excipients being chosen from Barley flour 13,41 g ggiare usually p y In these yp Maize fl 1M1 When added to animal feedstufisantibiotic 21,190 RP 322 525 22 makes it possible to achieve an inC e inthe Weight of Soya bean 11013;: 8 92 theanimal which is more rapid thanwith feedstuflfs devoid 5 Dehydrated hay f IL Yeast solids (whole cellsor autolysate) 2.33 Another aspect of the present invention thereforecom- Dried milk powder 2 68 prises animal feedstuffs, or concentratedmixtures for Sodium chloride im l f ing, c ntaining the antibiotic21,190 RP. Calcium i dose necessary to Produce f l Q P' Mineral elements(Mn, 1, and Z Q06 moting effect can naturally vary within quite widelirnits depending on the species of animals and on the nutri- Thlsfeedstufi contamsm addltlon: tional value of the feedstuffs themselves.In general terms, Wtamin A IU/kg. 4,000 it is sufficient for the feedrations made available to the Vltamill 3 IU/kg. 1,000 n mals o contain 1to 50 g. of antibiotic 21,190 RP h lin chloride mg./kg,11 5 per metricton of fcedstuff (and up to 80 per ri Rlboflaven m 2, ton for weanerfeedstuffs). The chicks are divided into 2 groups (4 coops per group,The antibiotic 21,190 RP can be contained as a uniform each groupcontaining 28 young hens and 28 young cocks). dispersion in completecomposite feedstuffs, at the above Group I receives the controlfeedstuff. Group II receives doses. l the control feedstuff to which theantibiotic 21,190 RP has It can be distributed in supplementaryfeedstuflfs to an been added at a concentration of 10 g. per metric tonof extent of 0.1 to 0.000l% by weight thereof, most frefeedstuff.quently with other additives such as vitamins and in- The resultsobtained are as follows:

I.--Average weight (in grams) On day 1 At 2 weeks At 4 weeks Young YoungYoung cocks cocks cocks Young Young yoi l r ifg Young Young yo iig YoungYoung oiiiig cocks hens hens cocks hens hens cocks hens hens Grou I Gm;11 3% ii 33 ii? iii l3? 2&3 232 3%? IL-GAINS IN WEIGHT AND CONSUMPTIONINDICES Group I (control) Group 11 Young Young cocks cocks and and YoungYoung young Young Young young cocks hens hens cocks hens hens Ida to2 eks:

Gain iii vseight (in g.) 151 144 148 165 156 160 Consumption index 1.56 1. 67 1. 62 1. 60 1. 62 1. 61 2 weeks to 4 weeks:

Gain in Wei ht (in 321 271 294 357 301 330 1 d Consumptlio n index 1.982.2 2.09 1.85 2.03 1.93

ay to 4wee s:

G i ht in 472 415 442 522 457 490 (7 rfi m ilgg lllliridex h i fil 1. 842.02 1. 93 1. 77 1. 89 1. 83 ififffffi-..3B?i?ifif?.?fif?ifi.ffffi 199199 199 119.9 119 119 23953813 315???511222352221?if???. 199 199 19999.1 99.5 99.9

EXAMPLE 3 Vitamin A IU 1,400,000 Vitamin D IU 401,500 The procedure ofExample 2 is followed, but using a xF y g1 tarmn mg ollowln com os1t1on:2 basic foodstuff having the f g p vltamm B3 "mg" 1,600 Vitamin B m 200Percent Vitamin B mg 3.1 Wheat flour 15 Vitamin C mg 1,000 Maize flourVitamin E mg 2,700 Fatty materials 0.8 zi i mg 6 25 9 8937a bean flour 7Folio acid mg F181 meal 35 Biotin m 10 Dried mllk powder Choline mg176,50 Distillers, solubles 2 The chickens are divided into 2 groups.Group I re- Mineral and vitamm complex 4 ceives the control feedstuif.Group II receives the con- This foodstuff contains 21% of digestibleproteins.

trol feedstuff to which the antibiotic 21,190 RP has been 40 added at aconcentration of 10 per metric ton of feed- The vitamin contents,calculated per kg., are as Stu-ff g follows: The results obtained are asfollows:

I.Average weight (in grams) On day 1 at 2 weeks at 4 weeks Young YoungYoung cocks cocks cocks and and and Young Young young Young Young youngYoung Young young cocks hens hens cocks hens hens cocks hens hens GroupI 36 35 36 206 194 200 639 582 610 Group II 36 35 35 212 203 207 662 620641 II. Gain in weight and consumption index Group I (control) Group IIYoung Young cocks cooks and and Young Young young Young Young youngcocks hens hens cocks hens hens 1 day to 2 Weeks:

Gain in weight (in g.) 159 164 176 168 172 Consumption index 1. 35 1.35 1. 35 1. 35 1. 32 1. 33 2 weeks to 4 weeks:

Gain in weight (in g.) 433 388 410 450 417 434 Consumption index 1.921.93 1.93 1. 83 1.90 1.86 1 day to 4 weeks:

Gain in weight (in g.) 603 547 574 626 585 606 Consumption index 1.76 1. 76 1. 76 1. 69 1. 73 1. 71 Gain in Weight (in percent relative tothe control) 100 100 100 100. 8 106. 9 105. 6 Consumption index (inpercent relative to the control) 100 100 100 96 98. 3 97. 1

We claim:

1. The antibiotic herein designated 21,190 RP, which possesses thefollowing characteristics: It is a white crystalline powder, melting at226228 C., soluble in water at pH 9 and in dimethylformamide, sparinglysoluble in methanol and ethanol, and practically insoluble in hexane andbenzene; it contains carbon, hydrogen, oxygen and chlorine and has theelementary composition: C=50.5%, H=6.6%, =38.4%, C1=4.5%; itsultraviolet spectrum in aqueous 0.1 N sodium hydroxide solution shows anabsorption maximum at 292 nm.

and an absorption minimum at 257 nm.

its infrared spectrum (determined with a mixture with potassium bromide)shows principal absorption bands as follows: 3,440 strong, 2,970 medium,2,930 strong, 2,900 shoulder, 2,840 shoulder, 1,735 strong, 1,715medium, 1,630 medium, 1,570 medium, 1,450 strong, 1,403 medium, 1,380strong, 1,350 medium, 1,335 shoulder, 1,305 shoulder, 1,248 strong,1,220 weak, 1,192 medium, 1,165 medium, 1,122 strong, 1,090 strong,1,060 strong, 1,035 very strong, 975 medium, 942 medium, 920 shoulder,900 weak, 865 medium, 848 weak, 828 weak, 815 weak, 775 medium, 730medium, 690 weak, 678 very weak, 655 weak, 630 shoulder, 612 weak, 570weak, 548 weak, 525 very weak, 510 very weak; and it optical rotation is2. Process for the production of the antibiotic 21,190 RP defined inClaim 1, which comprises culturing Streptomyces hygroscopicus, DS 23,230(NRRL 3576) under submerged aerobic conditions, commencing at a pHbetween 6.0 and 7.8, and at a temperature between 23 and 33 C. using anaqueous nutrient medium containing assimilable sources of carbon,nitrogen and inorganic substances until substantial antibacterialactivity has been imparted to the said medium, and isolating from themedium 21,190 RP formed during the culture.

3. Process according to Claim 2 in which the pH of the culture medium atthe beginning of the culture is between 6.5 and 7.5.

4. Process according to Claim 2 in which the temperature of the culturemedium is 25 to 30 C.

5. Process according to Claim 2 in which the culture medium is aeratedat a rate of 0.3 to 3 litres of air per litre of medium per minute.

6. Process according to Claim 2 in which 21,190 RP is separated from theculture medium by extraction with a water-immiscible organic solvent forthe said antibiotic selected from the class consisting of butanol,methylene chloride, ethyl acetate and methyl isobutyl ketone.

7. Process according to Claim 6 in which 21,190 RP is isolated from itssolution in the said water-immiscible organic solvent by concentratingthe solution under reduced pressure and precipitating the antibioticfrom the concentrate either by addition of hexane, or by cooling thesolution.

8. Process according to Claim 6 in which the extraction of theantibiotic from the culture medium is effected with the medium at a pHbetween 3 and 9.

9. Process according to Claim 8 in which extraction of the antibioticfrom the culture medium is eifected with the medium at about pH 7.

References Cited Miller: The Pfizer Handbook of Microbial Metabolites,McGraw-Hill Book Co., Inc. New York, N.Y., 1961, pp. 35, 40, 125, 126,128 and 580.

JEROME D. GOLDBERG, Primary Examiner US. Cl. X.R. -80

